Regioselective protein oxidative cleavage enabled by enzyme-like recognition of an inorganic metal oxo cluster ligand

Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K8[Cu2+(H2O)(α2-P2W17O61)], (CuIIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic CuIIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.


Supplementary Methods
General remarks. Unless otherwise noted, reactions were performed without any precautions against air and moisture. The cleavage reactions were performed in 1 mL aqueous solutions where the final concentrations of hen egg white lysozyme (HEWL), K8[Cu 2+ (H2O)(a2-P2W17O61)], (Cu II WD), or CuSO4 and sodium ascorbate (Asc) were 0.02, 0.05 or 2 and 1 mM, respectively. Stock solutions of HEWL (1 mM), Cu II WD or CuSO4 (10 mM) and Asc (200 mM) were prepared in 10 mM Tris-HCl, pH 7.5, unless otherwise noted. HEWL was purchased from Sigma-Aldrich. Sodium ascorbate was purchased from TCI and CuSO4 was purchased from Fluka Chemika. All chemicals were used without any further purifications. Hydrogen nuclear magnetic resonance ( 1 H NMR) spectra, and phosphorus nuclear magnetic resonance ( 31 P NMR) spectra were recorded on a Bruker Avance 400 spectrometer (400 and 376 MHz, respectively). Chemical shifts (δ) for hydrogens are reported in parts per million (ppm) downfield from tetramethylsilane propionic acid (TMSP-D4, 0.5 mM, 0 ppm) and are calibrated using the residual solvent peak in the NMR solvent (D 2 O: δ = 4.79 ppm). 31 (15 µL) were supplemented with 5 µL sample buffer (1M tris-HCl, pH 6.8 (2.25 mL), glycerol (5 mL), SDS (0.5 g), bromophenol blue (5 mg) and 1M dithiothreitol (2.5 mL) and heated at 95 °C for 5 min, followed by loading 10 µL of the resulting solution on the gel. Unstained low range (3.4 to 100 kDa) protein ladder (PL) was used as a molecular mass standard. An OmniPAGE electrophoretic cell was combined with an EV243 power supply (both produced by Consort, Turnhout, Belgium). Experiments were performed at 200 V for 2.0 h. Proteins in SDS-Tricine-PAGE gels were visualized with silver staining and an image of each gel was taken using a GelDoc EZ Imager (Bio-Rad, Hercules, CA). The percentage of the fragment bands compared to the total amount of protein in each lane was determined using the Bio-Rad Image Lab software Version 6.0.0 31 P NMR spectroscopy. A 1.5 mL centrifuge tube was charged with 200 µL of 20 mM Cu II WD stock solution, 20 µL of 1 mM HEWL stock solution, 10 µL of 200 mM Asc and 100 µL D2O and 780 µL buffer -10 mM tris-HCl (pH 7.4). The final concentration of Cu II WD was 2.0 mM, and of HEWL was 0.02 mM and 2.0 mM of Asc. The reaction mixture was homogenized using a vortex. Next, 500 μL of the reaction mixture was transferred into an NMR tube. The reaction was incubated for 1 day at 60 ⁰C. Then, the reaction was measured by 31  Circular dichroism spectroscopy (CD). Quartz cuvettes with 1.0 mm optical path length were used. The cuvette was loaded with 300 µL of HEWL (0.01 mM) and titrated with Cu II WD in presence and absence of Asc. All Solutions were prepared in 10 mM Tris-HCl buffer pH 7.5 and all measurements were done at 20 C. CD measurements were performed by using a JASCO J-810 spectropolarimeter. Far-UV wavelength scans were recorded from 180 to 300 nm. All the CD spectra were corrected for the background effect by subtracting the spectrum of the respective buffer solution from the spectrum of the protein.
nLC-MS/MS. After SDS-PAGE and Coomassie Brilliant Blue (CBB) staining and scanning (Supplementary Fig.   25), gel bands were excised, and in-gel digested with trypsin using the method described by Shevchenko et al. 32 The proteins were digested with proteomics-grade porcine trypsin at a ratio of 1:30. Peptides from in-gel digestions, were chromatographically separated using an UltiMate™ 3000 RSLC nano-liquid chromatographic system (nLC) (Thermo Fisher Scientific, Bremen, Germany), equipped with a 2-column set up: i) a trap column C18, 50 mm, and ii) an analytical column PepMap (C18, 15 cm × 75 μm, 3 μm particles, and 100 Å pore size). The mobile phases were (A) water (MS-grade) with 0.1% formic acid and (B) acetonitrile (MS-grade) with 0.1% formic acid. The gradient program was as follows: 0-0.5 min 95% A, 0.5-10 min 95-66% A, 10-15 min -66-0% A, 15-20 min 0% A, 20-23 min 5% A, with flow rate of 0.25 μL/min. This nLC system was coupled with an Orbitrap Exploris 240 high resolution mass spectrometer equipped with heated electrospray ionization source (Thermo Fisher Scientific, Bremen, Germany). Spray was generated with an integrated column emitter, with tip voltage set at 2. Proteins) database (downloaded on August 17, 2021 from http://www.thegpm.org/crap/). Oxidation (Met) and deamidation (Gln, Asn) were considered as variables, with carbamidomethylation (Cys) set as fixed in the PEAKS DB algorithm. In the PEAKS PTM algorithm, an PTM search was undertaken using a list of 313 PTM items. Up to two missed trypsin cleavages with non-specific cleavages at both ends of a peptide were allowed. Mass tolerances were set to ±10 ppm for parent ions and ± 0.02 Da for fragment ions. Protein filters were as follows: protein −10 lgP ≥ 20, proteins unique peptides ≥ 1, and "A" Score for confident PTMs identification of at least 50. Peptide filters were as follows: false discovery rate for peptide-spectrum matches < 0.5%; therefore, the resulting false discovery rate of the peptide sequence was lower than 1% (for more details check Smiljanic et al, 2019) 33 .
When identifying peptides with the PEAKS Studio X pro software package, all trypsin-derived peptides were taken into account, together with peptides generated by hydrolysis catalyzed by an enzyme of unknown specificity, to obtain all semi-tryptic and non-tryptic peptides. All semi-tryptic or non-tryptic peptides were extracted and overlapped with the peptides found in the control (Supplementary Table 1) in order to find peptides generated only by the action of Cu II WD/Asc. The sample preparation steps involved in peptide mapping are also sources of non-enzymatic PTMs. Alkaline pH, used during all steps of sample preparation, induces deamidation and disulfide bond scrambling and oxidation of methionine.
Sample treatment before analysis is necessary and can limit the information obtained. Because of the high tendency of oxidation at Cys residues during processing, reduction and alkylation is carried out. This therefore results in loss of most information about oxidant-mediated changes at Cys residues. Several studies have shown that reduction and alkylation can also decrease the levels of other modifications (e.g. 3-chloro-Tyr). Sequencing without alkylation and reduction can decrease sequence coverage.

Hydrolytic cleavage
The cleavage of HEWL via Cu II WD, in the absence of Asc, was monitored at pH 7.5, 60 °C for 3 days. Figure S14 shows that in presence of Cu II WD only three bands were produced with (11.5 kDa, 9.1 kDa and 7.6 kDa). Supplementary Figure 14, unambiguously, reveals that the bands produced from the cleavage of HEWL via Cu II WD is different from Cu II WD /Asc. This could be attributed to the difference in cleavage pathway since Cu II WD is most plausibly cleave protein through a hydrolytic pathway. Supplementary Fig. 15 shows two proposed pathways of the hydrolytic cleavage of the peptide bond based on literature. 2 In the first pathway ( Supplementary   Fig. 15-A) the metal could activate a coordinated hydroxyl or water which eventually will attack the amide carbonyl group. In the second one (Supplementary Fig. 15-B), the metal act as Lewis acid by activating an amide carbonyl towards nucleophilic attack by hydroxyl or water molecule from the solvent. Both mechanisms require at least one free coordination site at the catalyst in order to hydrolyze the peptide bond.  Supplementary Fig. 18 shows no change in the UV-Vis spectrum of Cu II WD before and after the reaction with HEWL/Asc, which indicates the stability of Cu II WD under reaction conditions. The increase in absorption intensity is due to the absorption of Asc (λmax= 265 nm) 3 Fig. 20 shows folded HEWL features spread between 6 and 10 ppm, in addition to peaks < 0 ppm.

1 H NMR spectroscopy Supplementary
As a control, 1 H NMR spectrum of HEWL in the presence of DTT and SDS was also recorded. DTT and SDS are known to cause effective protein denaturation, which leads to unfolding of the protein. From the data shown in Supplementary Fig. 20, most of the protein remains in its native form upon addition of Cu II WD, Asc or both.

Circular dichroism spectroscopy (CD)
The CD spectrum of HEWL in 10 mM tris-HCl (pH 7.4) at room temperature (Supplementary Fig. 21) shows a large minimum at  = 208 nm and a smaller minimum at  = 222 nm, both characteristic for -helical structure elements. 5 The minimum at  = 215 nm, characteristic of -sheet elements, is less pronounced because HEWL contains only a minor -strand region. 5,6 Supplementary Fig. 21 shows that Cu II WD does not change the overall shape of the HEWL CD spectrum but decrease the intensity of the negative signals at 208 ,215 and 222 nm. Upon addition of Asc (1 mM), a drastic change to HEWL CD spectrum was observed which in agreement with literature. 7 The minima at  = 215 and l=222 nm was almost lost while the peak at  = 208 nm was less affected. Addition of Cu II WD (0.05 mM) leads to restore minima at  = 215 and  =222 nm but decrease their intensity as well as minimum at  = 208 nm. Figure 21. CD spectra of HEWL/Cu II WD/Asc. CD evidence changes in HEWL (0.01 mM) secondary structure in the presence of Cu II WD (0.05 mM), Asc (1 mM) or both at 10 mM tris-HCl buffer pH 7.5 and 25  C. measurement was repeated two times with similar results on the same sample (technical replicates). Source data are provided as a Source Data file.

Mechanistic studies
To probe whether the Cu II WD mediated HEWL cleavage in the presence of Asc follows an oxidative pathway, several mechanistic experiments were conducted, based on schemes reported in the literature. 8,9 In the proposed mechanism, Asc reduces Cu II WD to Cu I WD, which in turn is able to reduce an O2 molecules from the air, leading to the formation of reactive oxygen species (ROS). The ROS presumably induce a radical formation on residues' side chains of HEWL prone to oxidation such as tryptophan, tyrosine, histidine and serine are ultimately cleaving protein through an oxidative pathway. 10,[11][12][13] Therefore, control experiments in the absence of Asc, or in the presence of radical scavengers, were carried. The nature of oxidant was probed in order to investigate the reaction mechanism, as discussed below in detail, and a reaction pathway coherent with the results is presented in (Supplementary Fig. 24).

Nature of reaction.
The different HEWL fragmentation pattern observed with and without Asc suggests that distinct cleavage reactions take place, supporting that an oxidative cleavage takes place in the presence of Asc. The hydrolysis of HEWL (0.02 mM) in the presence of only Cu II WD (2 mM) at pH 7.4 and 60 °C was much slower than in the presence of Asc, taking 3 days to produce bands with reasonable intensity in the SDS-PAGE analysis. 14, 15 Together, these result point to Cu II WD most plausibly cleaving the protein through a hydrolytic pathway in the absence of Asc. 2 In addition, they suggest that reaction undergoes a different pathway in the presence of Asc, most likely an oxidative one. [16][17][18]19,20,21 Radical scavengers. Overall conversion of the protein decreases in the presence of classical radical scavengers or under inert atmosphere, supporting the involvement of radical species in the reaction ( Table 2). Addition of mannitol (1 mM), a hydroxyl radical scavenger, 22 shortly after mixing Cu II WD /Asc with HEWL, led to a 47% decrease in the protein cleavage, while mixing mannitol with Cu II WD and HEWL before the addition of Asc completely inhibited the cleavage reaction. Further, the readily oxidizable tripeptide glutathione (GSH) 23 had no effect on the reaction when present in low concentration (0.02 mM), but a 78% inhibition was observed when an equimolar amount (1 mM) relative to Asc was used. 1 H NMR spectroscopy, Supplementary Fig. 22, showed the formation of GSSG, the oxidized form of GSH, which evidence the radical scavenging activity of GSH. Thiourea Role of ascorbate. The in situ formation of an ascorbyl radical was confirmed by electron paramagnetic resonance spectroscopy (EPR). The EPR spectra of Cu II WD solutions with and without HEWL were measured under the same conditions under which cleavage reactions were conducted and showed that an ascorbyl radical (Asc• -) was formed immediately after the addition of Asc in both cases (Figure 3). Accordingly, the formation of dehydroascorbic acid (DA) upon addition of Cu II WD to an Asc solution was also observed by 1 H NMR spectroscopy ( Supplementary  Fig. 23 (Supplementary Fig. 11), corroborating the oxidative character of the reaction, and pointing out the crucial role of oxygen in the mechanism. In general, the reduction of oxygen has been proposed to afford reactive oxygen species like O2 •-, O2 2or HO • , which can be protonated in solution to generate peroxides that could further react. However, no cleavage was detected when HEWL was treated with Cu II WD /H2O2 (0.05 mM/1 mM) instead of the usual Cu II WD/Asc combination (Supplementary Fig. 12), strongly suggesting that any H2O2 eventually formed in solution is not responsible for the cleavage observed. This contradicts common mechanistic proposals stating H2O2 as an intermediate in similar reactions, 25 though the reactivity of Cu II complexes towards H2O2 largely depends on the redox potential of the complex, which may vary widely due to the ligands in the coordination sphere of Cu II , and/or the reaction conditions. 26,27,28 Together, these results suggest a mild oxidant species formed through the reaction of Cu I WD and O2 might be enabling HEWL oxidative cleavage. 29,30 Importantly, this would also account for the fact that the reaction is not inhibited Asc's known radical scavenger ability, 31